Abstract Significance: Intracellular delivery of nucleic acids to immune cells is an important component of many current and anticipated cellular therapies, including chimeric antigen receptor T Cell (CAR-T) therapies for the treatment of cancer. These therapies use immune cells called T cells, either from the patient themselves, or a healthy donor, and genetically engineer them to kill the tumor cells. The genetic engineering involves direct delivery of nucleic acids to these cells, which is challenging as current delivery methods either lack efficiency, are very damaging to cells, or both. This becomes a critical concern for strategies that either seek to perform multiple, sequential deliveries or aim to generate a large number of cells (1010) for development of an off-the- shelf therapeutic. OpenCell Technologies (OCT) has developed a proprietary technology, POROS?, to deliver macromolecules such as DNA, RNA and protein to a wide variety of cell types. POROS uses acoustic force to push cells through an array of nozzles one cell at a time thus creating shear force to porate cells. In this SBIR project, OCT will expand the capabilities of its POROS platform by developing higher through-put POROS devices. Background: CAR-T therapy is a rapidly emerging therapy for the treatment of cancer, with two companies recently receiving FDA approval for their first CAR-T therapies. The existing therapies involve using the patient?s own cells and engineering them to attack the tumor cells. This engineering is performed on a case-by- case basis and the methods are costly and time-consuming. In order to make CAR-T therapies available to more patients in a cost-effective, safe and timely manner, a new approach needs to be developed. Current research is addressing this need by engineering healthy donor T cells to be used in CAR-T therapy. However, methods and technology need to be developed to support this new approach, which will involve multiple gene- editing steps and on a much larger scale. Approach: In this Phase I feasibility study, OCT will demonstrate that its delivery technology, POROS, can be expanded to treat a larger number of cells to enable CAR-T development. In this project, we will develop a prototype POROS-midi device, capable of treating 108 cells in under 10 minutes, and demonstrate gene editing in T cells as a proof-of-concept experiment. While the POROS-midi device development will serve as a feasibility study for development of a large, all-in-one, GMP-compliant POROS-giga device, POROS-midi will also be a salable unit of its own with anticipated applications in personalized medicine and research and development.